Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Summary Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)-α-hydroxymethyltyrosine in position 1 and (R)- and (S)-α-hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that α-hydroxymethyl substitution of amino acid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

Free amino acids in the seeds of Acacia species

Free amino acids in the seeds of 80 species of Acacia have been determined. In addition to confirming the known difference in seed amino acid ‘patterns’ shown by species of the series Gummiferae on the one hand and species of the series Phyllodineae and Botryocephalae on the other, we have found that species of the series Vulgares Benth. show two additional ‘patterns’. One of these is common to some of the Afro-Asian species of the series which have been analysed and the other is common to species from America and Africa. One of the ‘marker’ amino acids of the Afro-Asian group is the neurotoxic lathyrogen α-amino-β-oxalylaminopropionic acid. The possible taxonomic and phylogenetic significance of these results is discussed, with special reference to five species A. albida, A. confusa, A. heterophylla, A. coulteri and A. kauaiensis.     

Proteinase inhibitors from Erythrina corallodendron and Erythrina cristagalli seeds

Eight and five proteinase inhibitors were purified from Erythrina corallodendron and E. cristagalli seeds, respectively, by gel filtration followed by ion exchange chromatography on DEAE-cellulose and DEAE-sepharose. Each inhibitor consists of 161–163 amino acids (M_r 18 000) including four half-cystine residues and resembles the Kunitz-type proteinase inhibitors. The N-terminal amino acid sequence of trypsin inhibitor DE-7 from E. corallodendron seed resembles those of other Erythrina species. For the other inhibitors no free N-terminal amino acid was found. DE-1,-2,-3,-4 and -5 from the seed of E. corallodendron contain potent inhibitors for α-chymotrypsin and they have practically no action on trypsin. From the same seed, inhibitors DE-6, -7 and -8 strongly inhibit trypsin and also inhibit α-chymotrypsin to varying degrees. From the seeds of E. cristagalli, inhibitors DE-1 and -8 inhibit trypsin strongly and DE-2, -3 and -4 are strongly inhibitory for α-chymotrypsin. On summarizing the inhibitor characteristics of the Kunitz-type proteinase inhibitors from the seeds of eight different species of Erythrina, it was obvious that there is a relationship between the alanine content of the inhibitors and their activities. A high alanine content is associated with potent α-chymotrypsin activities and low alanine content with strong trypsin activities.     

Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)--hydroxymethyltyrosine in position 1 and (R)- and (S)--hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that -hydroxymethyl substitution of aminoacid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

Non-protein amino acids of Acacia species and their effect on the feeding of the acridids Anacridium melanorhodon and Locusta migratoria

The leaves of Acacia species have been found to contain homoarginine, pipecolic acid and 4-hydroxy-pipecolic acid. The nymphs of the tree locust Anacridium melanorhodon, which feed on the leaves of Acacia species, were not inhibited from feeding on palatable media containing concentrations of these amino acids equivalent to, or greater than, those found in the leaves. The graminivorous Locusta migratoria was more sensitive to these compounds, inhibitory effects being observed at concentrations comparable to those found in the leaves. The inhibitory effects of mixtures of homoarginine and pipecolic acid were additive in A. melanorhodon but not in L. migratoria. Three of the non-protein amino acids found in the seeds of Acacia species, 2,3-diaminopropionic acid, 2-amino-3-acetylaminopropionic acid and 2-amino-3-oxalylaminopropionic acid, were more effective inhibitors of feeding in Anacridium than were the leaf amino acids.     

4-Carboxy-4-hydroxy-2-aminoadipic acid and other acidic amino acids in Caylusea abyssinica

Two diastereoisomers of 4-carboxy-4-hydroxy-2-aminoadipic acid have been isolated from leaves and inflorescences of Caylusea abyssinica. Green parts of the plant also contain appreciable amounts of the two diastereoisomers of 4-hydroxy-4-methylglutamic acid, 3-(3-carboxyphenyl)alanine, (3-carboxyphenyl)glycine, 3-(3-carboxy-4-hydroxyphenyl)alanine, (3-carboxy-4-hydroxyphenyl)glycine and in low concentration 2-aminoadipic acid, saccharopine [(2S, 2′S)-N⁶-(2-glutaryl)lysine] and some γ-glutamyl peptides. The acidic amino acids were separated from other amino acids on an Ecteola ion exchange column with M pyridine as eluant.     

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.     

Transformation of 3-(3-carboxyphenyl)alanine in iris species An example of diversity in catabolism of secondary plant products

3-(3-Carboxyphenyl)-DL-[2-¹⁴C]alanine has been incorporated into four species of iris. In all species extensive metabolization takes place. In Iris × hollandica, in which both the alanine derivative and 3′-carboxyphenylglycine occur, the products identified are the glycine derivative, 3′-carboxyphenylacetate acid, 3′-carboxymandelic acid, and 3′-carboxyphenylglyoxylic acid. In I. sanguinea, in which the alanine and glycine derivatives also occur, the products identified are the glycine and acetic acid derivatives but the major product is 3-(3-hydroxymethylphenyl)alanine, a naturally occurring amino acid in this species. In I. tectorum, in which only the carboxy-substituted alanine derivative occurs, the products identified are the acetic acid and glyoxylic acid derivatives. In I. pallida, not containing any of the meta-substituted amino acids, the products identified are again the acetic acid and glyoxylic acid derivatives. The results have been further substantiated by incorporation of labelled 3′-carboxyphenylacetic acid and 3′-carboxymandelic acid into I. × hollandica and I. sanguinea.The results demonstrate three different metabolic patterns for the alanine derivative and confirm previous results on the pathway from the alanine to the glycine derivative. Furthermore, the results may be of significance for the elucidation of the catabolism of phenylalanine.     

Delta-6 desaturase makes a different fatty acid profile of seed oil in Iranian borage (Echium amoenum Fisch. and Mey.)

Iranian borage (Echium amoenum) from Boraginaceae is a valuable medicinal plant native to Iran and Syria. We determined fatty acid profile and individual fatty acid contents in E. amoenum seed oil using gas chromatography. Nevertheless, the cumulative amount of ω-6 and ω-3 fatty acids in E. amoenum (78.5 %) was in great accordance with those of other species of Echium, Stearidonic acid (SDA, C18:4ω3) and gamma-linolenic acid (GLA, C18:3ω6) constituted only 9.7 % of seed oil in this plant. This observation elucidated the weak activity of delta-6 desaturase (D6DES) in E. amoenum compared with D6DESs of other species. As D6DES enzyme is responsible for converting linoleic acid (LA, C18:2ω6) and alpha-linolenic acid (ALA, C18:3ω3) to GLA and SDA, we isolated coding sequence of D6DES gene and characterized primary structure of the translated protein to probably find some evidences explaining the weak activity of D6DES enzyme in E. amoenum. Gene sequence from E. amoenum showed a high identity of 94–96 % with the other Echium species and the amino acid homology increased by 98 %. All the expected signatures including cytochrome b5 domain and three conserved histidine-rich motifs were found in the translated amino acid sequence. Protein alignment revealed that all the conserved motifs in D6DES sequence from E. amoenum are coincident with its counterparts from other Echium species. However, secondary structure of the enzyme deduced from its primary structure using computational simulation represented obvious differences with D6DES proteins of the other species.     

Variability for protein and oil quality in Lupinus albus

Amino acid composition and fatty acid composition were determined on seed samples of a range of white lupin (Lupinus albus) cultivars and accessions grown in either of two environments.Variability between genotypes was found for lysine, arginine and glutamic acid content, but not for the concentrations of other amino acids. The deficiency in sulphurcontaining amino acids, typical of legume proteins, was evident, with methionine and cyst(e)ine totalling only 2.2% of the protein. Variability was limited, indicating that improvement by breeding would be impracticable. Lupinus albus differed slightly from other lupin species in amino acid composition, having higher levels of threonine, tyrosine and isoleucine, but a lower level of glutamic acid than both L. angustifolius and L. luteus. Four low-alkaloid lines of L albus each had higher lysine content than the high-alkaloid line, but ‘Kiev Mutant’, despite earlier claims, had a lysine level no higher than the other three low-alkaloid lines.Fatty acid composition of the seed oil varied considerably between genotypes. Oleic acid ranged from 43.6 to 54.4% and linolenic acid from 6.7 to 15.2%, these two fatty acids being negatively correlated at one site. Linoleic acid content varied between 17.2 and 26.9% and was not correlated with other fatty acids. Total oil content averaged 9.6% with little variability between lines.It is concluded that, relative to other lupin species, L. albus has a more favourable amino acid profile for its utilisation in cereal-based diets for animals, particularly if the energy source is wheat, which is deficient in threonine. The higher oil content would be an important energy benefit to such diets and may allow their protein/energy balance to be maintained at higher levels of incorporation of L. albus seed meal than is possible with other lupin species.     

Amino acids,sugars and sterols of some Mediterranean brown algae

Free amino acids, amino sulfonic acids, sugars and sterols have been examined and quantitatively determined in 10 brown seaweeds. Acidic amino acids and their amides are the main components of the amino acid fraction. Cysteinolic acid, taurine and its N-methyl derivatives have been identified in most of the species examined. In all the algae, mannitol is present, sometimes in very large amounts. The sterol fractions of all the species contain fucosterol, cholesterol and 24-methylenecholesterol; minute amounts of 22-dehydrocholesterol and 24-methylcholesta-5, 22-dien-3β-ol have also been frequently detected.     

Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.     

Leucine specific transfer ribonucleic acids and synthetases in the cotyledons of mature and germinating pea seeds

Changes in isoaccepting species of tRNA^Leu were determined in germinating pea seedlings and in developing pods. Leucine specific transfer ribonucleic acids of pea cotyledons can be fractionated into four isoaccepting species by reversed-phase chromatography (RPC-5) on a Plaskon column. In contrast, only two species of tRNA^Leu were observed in developing seed pods. Leucyl-tRNA synthetase purified by ammonium sulfate precipitation and DEAE cellulose column chromatography retained the full range of specificity towards all four tRNA^Leu species of pea cotyledons. This partially purified pea cotyledon enzyme could be further separated on a hydroxylapatite (HA) column into two peaks of leucyl-tRNA synthetase activity. Enzyme 1 is dominant in seed pods while 2 is predominant in cotyledons. Enzymes 1 and 2 from cotyledons were examined for the amino acid acceptor activity of twelve different amino acids. Both these fractions showed less than 3% acceptor activity for eleven other amino acids as compared to leucine-tRNA synthetase activity. Preliminary characterization of enzyme 2 from cotyledon, by isoelectric focusing and polyacrylamide gel electrophoresis indicates at least three subspecies.     

Sequence Diversity of the Bacillus thuringiensis and B. cereus Sensu Lato Flagellin (H Antigen) Protein: Comparison with H Serotype Diversity

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis, in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans, B. subtilis, and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides, B. weihenstephanensis, B. cereus, and B. thuringiensis, did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.     

Effectiveness of the fatty acid and sterol composition of seeds for the chemotaxonomy of Coffea subgenus Coffea

The chemotaxonomic relationships between Coffea (subgenus Coffea) species have been poorly studied to date and the compounds tested so far - chlorogenic acids, diterpenoids and purine alkaloids - did not enable the establishment of phylogenetic relationships analogous to those revealed by chloroplast and nuclear DNA studies. In the present study, the relationships between African Coffea species were assessed on the basis of their seed lipid composition. Fatty acids and sterols were determined in 59 genotypes belonging to 17 distinct Coffea species/origins. Principal Component Analysis of fatty acid and sterol data enabled easy identification of the few species for which one or several compounds could serve as a quantitative signature. Hierarchical Clustering classified the Coffea species in seven groups with both fatty acids and sterols. However, while groupings based on seed fatty acid composition showed remarkable ecological and geographical coherence, no phylogeographic explanation was found for the clusters retrieved from sterol data. When compared with previous phylogenetic studies, the groups deduced from seed fatty acid composition were remarkably congruent with the clades inferred from nuclear and plastid DNA sequences.     

Free amino acids and γ-glutamyl peptides in seeds of Fagus silvatica

The contents of amino acids and peptides have been investigated in seeds of Fagus silvatica L. (beechnuts). In addition to the common amino acids, the following compounds have been isolated and identified: 4-hydroxyproline (probably the cis-l-isomer), N⁵-acetylornithine, 3-(2-furoyl)-l-alanine, methionine sulfoxide (probably an artefact), pipecolic acid (probably partially racemized d-isomer), l-willardiine (with a small amount of the d-isomer), N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid, N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid, 2(S),5(S),6(S)-5-hydroxy-6-methylpipecolic acid, 2(S),5(R),6(S)-5-hydroxy-6-methylpipecolic acid, γ-glutamylalanine, γ-glutamylglutamic acid, γ-glutamylisoleucine, γ-glutamylleucine, γ-glutamylmethionine sulfoxide (probably an artefact), γ-glutamylphenylalanine, γ-glutamyltyrosine, γ-glutamylvaline, glutathione, γ-glutamylwillardiine, and γ-glutamylphenylalanylwillardiine. γ-Glutamylphenylalanine and willardiine are the dominating components of the amino acid fraction.The isolations were performed by use of ion exchange chromatography, taking advantage of the different pK-values of the amino acids, mainly on acid resins in the 3-chloropyridinium form with aq. 3-chloropyridine as eluant and on basic resins in the acetate form with aqueous acetic acid as eluant. These methods in combination with preparative paper chromatography have permitted the isolation and identification of compounds present in amounts as low as 1/6000 of the dominant ninhydrin-reactive component. The implications of the occurrence of this large variety of compounds in the Fagaceae are briefly discussed.     

Triterpene acids from Salvia and Teucrium species

The pentacyclic triterpene acids ursolic acid, oleanolic acid, micromeric acid, maslinic acid and 3-epi-maslinic acid have been isolated from several Salvia and Teucrium species.     

4-Hydroxyisoleucine from seed of Trigonella foenum-graecum

The principal free amino acid present in seed of Trigonella foenum-graecum has been isolated and identified as (2S, 3R, 4R)-4-hydroxyisoleucine. This compound has not been reported previously as a constituent of higher plants, but it is a component of the toxic peptide, γ-amanitin, produced by Amanita phalloides. The (2S, 3R, 4R)-isomer lactonizes readily under acidic conditions, whilst strong acid causes partial epimerization. The (2R, 3R, 4R)-isomer forms a minor component of Trigonella seed. The 4-hydroxyisoleucine content of fenugreek increases during the growth of seedlings and plants, and ¹⁴C-isoIeucine was used effectively as a biosynthetic precursor.